The separation of the fluid fraction, whether serum or plasma, from blood is of great importance in the clinic. Blood is made up of two fractions: blood cells, and the liquid in which they are suspended, blood plasma (“plasma”), a proteinaceous fluid. The important cell types encountered in blood are the red blood cells, the white blood cells and platelets although other cell types may be detected as well. Blood serum (“serum”) is the fluid remaining after the removal of fibrin clots and cells.
Although whole blood is obtained relatively easily and in principle is capable of providing valuable information, its use is limited by problems in handling it for use in reliable rapid diagnostic assays. For example, if a diagnostic assay is based on a colorimetric reaction, hemolysis of red blood cells introduces errors. Even when the readings are not affected by colors contributed by hemolysis, the presence of cell lysates and its contribution variable recovery of the fluid fraction is a source of error. It is therefore desirable to reproducibly separate cellular components of whole blood to obtain a fluid fraction that can then be used in downstream applications.
The ability to measure a wide variety of physiologically active substances, both naturally occurring and synthetic, has assumed increasing importance as an adjunct to both diagnosis and therapy. While for the most part such assays require clinical laboratory determinations, there is an increasing awareness of the importance of being able to conduct assay determinations in a physician's office or in the home. These latter environments typically require that the assay have a simple protocol and be relatively free of sensitivity to small changes in the conditions under which the assay is carried out. Importantly, inaccurate measurements of reagents and sample should, whenever feasible, be avoided.
It is desirable that such devices and assays are fast, easy to use with few or no complicated procedures, readily readable, accurate and capable of being manufactured in mass quantities at a low cost (per unit). These goals usually require that the devices be amenable to manufacture using automation and be sufficiently robust for the handling required therefor.
The use of whole blood in the case of certain rapid diagnostic assays creates special problems. For example, if the diagnostic assay is based on a calorimetric reaction, turbid or colored solutions, such as from red blood cells, disturb the readings. It is therefore desirable to separate cellular components of whole blood to obtain the fluid fraction that can then be used in downstream applications. In particular, it is desirable to separate cellular components from the fluid fraction of whole blood in the absence of hemolysis because hemolysis causes components of red blood cells to be released into the fluid fraction and potentially interfere with downstream applications.
Attempts have been made to develop rapid diagnostics for the direct use of whole blood. For example, test papers coated with semi-permeable membranes to prevent the contact of larger components of the sample to contact the test paper have been developed (see, e.g., U.S. Pat. No. 3,092,465). Another example is the use of swellable films into which only the dissolved components of the blood, but not the erythrocytes, can penetrate (see Federal Republic of Germany Patent Specification No. 15 98 153). However, higher molecular weight components of the blood, for example lipids or substrates bound to serum proteins, for example bilirubin, cannot be determined in this way because they are also not able to penetrate into the film or to pass through the semi-permeable membrane. A disadvantage of certain filter systems is that blood penetrates through the membrane filter very slowly.
A conventional manner of separating the fluid fraction from erythrocytes is centrifuging. However, especially in the case of using small amounts of sample, such as 50 microliters or less, this gives rise to problems and the separation of supernatant and precipitated cellular components. Further, conventional methods require more handling time by the doctor, nurse, technician, or tester. Such additional handling is generally undesirable for efficiency as well as hygienic reasons. Moreover, in some point of care situations, a centrifuge may not be available.
Therefore, it is desirable to create a device to reduce the amount of handling required to separate blood fluids from cellular components of whole blood and, in particular, rapidly and effectively separate the fluid fraction from cellular components of small volumes of blood.
It is known that lymphocytes and leukocytes can be separated from blood when blood is filtered through a layer of synthetic resin fibers with an average fiber diameter of 5 to 20 micrometer in the case of separating lymphocytes, and of 3 to 10 micrometer in the case of separating leukocytes (see, e.g., Federal Republic of Germany Patent Specifications Nos. 29 08 721 and 29 08 722). However, since the erythrocytes can pass through the filter along with the plasma, these filters are not suitable for obtaining plasma because the erythrocytes can interfere with the downstream application, such as a calorimetric test.
Separators that separate the fluid fraction from blood cells are known in the art. The U.S. Pat. Nos. 4,477,575 and 4,816,224 issued to Vogel et al. on Oct. 16, 1984 and Mar. 28, 1989 respectively. (“the Vogel patents”) were the first to disclose the use of glass fibers having density of 0.1 to 0.5 gm./ml with an average fiber diameter of 0.2 to 5 microns for separating the fluid fraction from blood.
Additional devices for separating the fluid fraction from whole blood that involve a glass fiber-based filtration mechanism are described, e.g., U.S. Pat. No. 4,696,797, issued Sep. 29, 1987, to Kelton. To facilitate the handling of small volumes of blood, e.g., 50 microliters or less, dye indicator systems as described, e.g., in U.S. Pat. No. 6,544,474, issued Apr. 8, 2003, to Douglas, were developed. Blood separation devices are described in U.S. Pat. No. 5,135,719, issued Aug. 4, 1992, to Hillman et al. that include a glass microfiber filter or filters with agglutinin for separating cellular components, e.g., erythrocytes, from the fluid fraction of whole blood. Agglutinin promotes the aggregation of blood cells and thereby improves the filtration process. The sole driving force for the movement of plasma from the filter to a downstream application is capillary force provided by a tubular capillary. A potential drawback of these glass fiber-based separators is that the separation of blood cells from whole blood often does not occur without hemolysis. Due to the hemolysis, certain components of erythrocytes contaminate the recovered fluid fraction, which may interfere with downstream applications.
In an effort to reduce hemolysis during the separation process, blood separators were developed with a layer of glass fibers, wherein the glass fibers are coated with, e.g., polyvinyl alcohol and/or wherein the layer contains an erythrocyte-aggregating substance. Such a device is described in U.S. Pat. No. 5,262,067, issued Nov. 16, 1993, to Wilk et al.
Certain improvements to the glass fiber-based separation method were described, e.g., in U.S. Pat. No. 5,435,970, issued Jul. 25, 1995, to Mamenta et al., which discloses devices for separating blood cells from biological fluids using a non-absorbent, porous unitary support, which can be made from glass fiber, and a blood cell binding composition, such as lectin. Similarly, U.S. Pat. No. 6,008,059, issued Dec. 28, 1999, to Schrier et al., describes the separation of the liquid portion of blood from the cellular components of whole blood using a pad of porous material. The pad of porous material is permeable to the liquid portion of blood but capable of trapping the cellular components of blood. The pad of porous material is further described as typically containing a binder for the cellular components of blood. The blood cell binding composition improves the effectiveness and efficiency of the blood separation process.
Many attempts for separating the fluid fraction from blood use multiple layers of filters and paths in attempts to permit flows around or past regions that have become clogged due to red blood cells. None of the known methods teach demonstrably gentle methods for trapping red blood cells in a region where the blood sample is applied with little to no migration of red-blood cells while the fluid fraction is separated efficiently.
U.S. Pat. No. 5,916,521 issued to Bunce et al. on Jun. 29, 1999 (“the '521 patent”) discloses the use of a filter comprising sheet like capillary forms such that a first capillary form has a larger pore size for allowing some particulate matter to pass and is laterally connected to a second capillary form having a smaller pore size for trapping all particulate matter. Within each sheet, the pore sizes are substantially isotropic. The '521 patent also teaches lateral flow of blood to open pores if pores in a transverse path are blocked resulting in a progressively spreading region impregnated with red blood cells, both due to lateral flow within the filter and around the clogged pores at the site of application of blood.
In addition to glass fiber filters, polysulfone resins have also been used in blood separators. Polysulfone resins are extremely chemically resistant and also have some desirable mechanical properties. They, however, must be converted to a micro-porous structure, either in the form of a sheet stock or as fibers, to function as blood separators. Presently, two companies offer polysulfone based blood separators, namely Pall Corporation and Prime Care (Holland). These blood separators are in a sandwich form with separate collector pads. These separators also do not readily release the fluid fraction. In another aspect, polysulfone based materials also require multiple steps during manufacturing and are expensive compared to glass fiber papers.
U.S. Pat. No. 4,216,280, issued Aug. 5, 1980, to Kono et al. (the “Kono patent”) discloses glass fiber paper based battery separator mediums and methods for making them from combinations of glass fibers having different diameters. The Kono patent also discloses battery separator mediums having, in addition to glass fibers, synthetic resin fibers such as polyester, polypropylene, or acrylonitrile fibers that are uniformly dispersed within the glass fibers. The Kono patent actually discloses making glass fiber papers that are suitable for retaining fluids as opposed to separating a fluid fraction from a whole blood sample.
In summary, the known art employs various combinations of pore-sizes, hydrophobic surfaces, glass fibers and the like in the quest for a device suitable for separation of the fluid fraction from blood. The known art teaches the use of thin fibers, typically having a diameter less than five microns and preferably around or below one micron. The methods also rely upon speedy removal of any recovered fluid fraction since red blood cells eventually reach and increasingly contaminate the recovered fluid fraction, both via direct contamination and due to hemolysis over time.
Significantly, the known methods and devices are not amenable to automation since, the most effective separation medium used therein, glass fibers, are brittle and difficult to handle. Thus cutting the material into specific and accurate shapes and packaging thereof, often requires significant amount of manual labor rather than automated handling.